Structure of a ribozyme catalytic center

描述（由申请人提供）：对于真核细胞中的大多数基因，初级转录物的剪接是由兆道尔顿分子机器剪接体进行的，剪接体由100多种蛋白质和5种小核RNA（snRNA）组成。然而，这种剪接反应也是由II组内含子RNA酶（核酶）进行的，而不需要蛋白质。这种核酶与剪接体在结构和机制上有着惊人的相似性，这使得它们成为研究剪接基础生物化学的一个有吸引力的模型。现在已经确定结构域1至3（D123）、结构域5（D5）和外显子底物形成II组内含子催化所必需的最小活性位点。活性部位组分的唯一已知结构是孤立的D5。然而，单独的D5不能模拟活性催化构型。因此，在D123和底物的背景下检查D5的结构对于推进我们对催化的原子基础的知识至关重要。为此，我们已经重建了第二组内含子Pylaiella littoralis成一个新的三方核酶系统，有一些重要的区别，从以前的模型系统，使其成为一个有吸引力的目标结构研究。我们推测D5和底物的内部凸起形成催化位点的关键部分。我们将应用强大的生物物理和生物化学工具，以获得详细的结构洞察这种核酶的最小催化核心。我们的总体具体目标是：1.确定D5凸起缺失突变体-D5 A25 δ的催化缺陷的分子基础。2.使用核磁共振、突变和紫外线交联绘制存在和不存在底物情况下D5凸出突变体和D123之间的结合界面3。为了获得最小活性位点的原子视图并鉴定对结合和催化重要的特征，使用NMR确定与D123（和/或D123的片段）和底物结合的D5和D5凸起突变体的结构，和4.用生化分析鉴定其结构。重要性：我们预计，这些研究的完成将提供一个原子框架，解释广泛的生化和遗传数据组II内含子，以扩大我们的理解催化，这些可能提供的第一个证据的细节RNA相互作用在细胞剪接，并建议新的方法来开发组II内含子作为潜在的核酶治疗。

DESCRIPTION (provided by applicant): For most genes in eukaryotic cells, splicing of the primary transcript is undertaken by a megadalton molecular machine, the spliceosome, consisting of over 100 proteins and five small nuclear RNAs (snRNAs). However, this splicing reaction is also carried out by the Group II intron RNA enzymes (ribozymes) without the need of proteins. Such ribozymes share striking structural and mechanistic similarities to the spliceosome, which makes them an attractive model for studying the basic biochemistry of splicing. It is now well established that domains 1 through 3 (D123), domain 5 (D5) and exon substrates form the minimal active site essential for catalysis by group II introns. The only known structure of the active site component is D5 in isolation. Yet D5 alone cannot mimic the active catalytic configuration. Examining the structure of D5 in the context of D123 and substrates is therefore critical for advancing our knowledge of atomic basis for catalysis. To this end we have reconstructed a group II intron from Pylaiella littoralis into a novel tripartite ribozyme system that has some important difference from previous model systems and that makes it an attractive target for structural studies. We hypothesize that the internal bulge of D5 and substrate form a key part of the catalytic site. We will apply powerful biophysical and biochemical tools to obtain detailed structural insight into the minimal catalytic core of this ribozyme. Our overall specific aims are: 1. Determine the molecular basis for the catalytic deficiency of a D5 bulge deletion mutant-D5A25delta. 2. Map the binding interface between D5 bulge mutant and D123 in the presence and absence of substrates using NMR, mutagenesis, and UV crosslinking 3. To obtain an atomic view of the minimal active site and to identify the features that are important for binding and catalysis, determine the structure of D5 and D5 bulge mutant bound to D123 (and or fragments of D123) and substrate using NMR, and 4. Validate the structure using biochemical assays. Significance: We anticipate that completion of these studies will provide an atomic framework for interpreting the extensive biochemical and genetic data on group II introns to extend our understanding of catalysis, these might provide the first evidence for the details of RNA interactions in cell splicing, and suggest novel approaches to developing group II introns as potential ribozyme therapeutics.